017.Tran Van Chieu_htt (132-140)

Tp ch Khoa hc 2010:15b 132-140 Trng i hc Cn Th

132

NH HNG CA INDOLE ACETID ACID (IAA) DO VI KHUN AZOSPIRILLUM TNG HP LN S PHT

TRIN CA R LA TRNG IU KIN NH LI

Trn Vn Chiu1 v Nguyn Hu Hip2

ABSTRACT

Three Azospirillum lipoferum strains isolated and identified by Polymerase Chain

Reaction (PCR) technique were used to test their ability of synthesis of Indole Acetic acid

(IAA) and their effects on the growth of rice root cultivated in the greenhouse.

The results showed that all of three strains R7b1, R8b2 and R29b1 could synthetized high

amout of IAA than the control in the medium without Tryptophan. Azospirillum lipoferum

strain R29b1 could synthesized the highest amount of IAA (19,9g/ml) after inoculating 4

days. These amount of IAA supported the growth of rice root length and the amount of

lateral root grown in the green house. After inoculating 28 days, the rice root length were

2.03 times compared to the control.

Keywords: Azospirillum lipoferum, Indole Acetic acid (IAA), tryptophan, rice root,

green house

Title: Effect of Indole Acetic acid (IAA) synthesized by Azospirillum on the growth of

rice root grown in the green house

TM TT

Ba dng vi khun Azospirillum lipoferum phn lp c t r la, nhn din bng k thut PCR, c chn kho st kh nng tng hp IAA v nh hng ca chng ln s pht trin ca r la trong iu kin nh li.

Kt qu th nghim cho thy, c ba dng R7b1 R8b2 v R29b1 u tng hp c lng IAA nhiu hn i chng trong mi trng nui khng c Tryptophan. Dng R29b1 tng hp c lng IAA nhiu nht 19,9g/ml vo ngy th 4 sau khi chng. Lng IAA ny gp phn lm tng chiu di r la v tng s lng r ph trong th nghim nh li. Chiu di r la tng 2,03 ln so vi i chng khi c chng dng R29b1 sau 28 ngy.

T kha: Azospirillum lipoferum, Indole Acetic acid (IAA), tryptophan, r la, nh li

1 T VN

Vit Nam ang l nc ng th hai trn th gii v sn lng la go xut khu (http://www.fita.org/countries/vietnam.html) (Truy cp vo thng 11/2009). Trong , ng bng sng Cu Long ng vai tr rt quan trng (ng gp 90% lng go xut khu ca Vit Nam) (http://www.tapchicongsan.org.vn/print.asp) (truy cp ngy 11/02/2009).

Mun tng nng sut cho cy la, ngi nng dn s dng mt lng ln phn ha hc, trong c phn m. Tuy nhin, bn cnh mt tch cc, lng phn ha

1 Nghin cu sinh chuyn ngnh Vi sinh vt 2 Vin Nghin cu v Pht trin Cng ngh sinh hc, Trng i hc Cn Th


133

hc tha gy nhim mi trng, lm cho con ngi, c bit l tr em, v gia sc b ng c. Thm vo , phn ln phn ha hc u c nhp khu vi chi ph rt cao.

gii quyt vn , xu hng s dng phn sinh hc cha cc loi vi khun c trong t ang c quan tm. Mt s dng vi khun thuc ging Azospirillium v ang c cc nh khoa hc trn th gii quan tm nghin cu. Ngoi kh nng c nh N cho cy trng, Azospirillum cn c th tit ra nhng kch thch t tng trng nh: IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), ABA (Abscisic acid) v cytokynins (Bashan v Levanony, 1990). Nhng kch thch t lm tng chiu di r, tng th tch r v s lng r. T , chng lm tng kh nng hp thu khong cht v nc, nh , tng kh nng sinh trng v pht trin cng nh tng nng sut ca cy (Okon and Kapulnik, 1986). Ngoi ra, chng cn gip cho cy chng chu c iu kin kh hn. Kolb v Martin (1985) phun dch vi khun A.brasilense FT-326 trn r ca Beta vulgaris lm cho r mc di hn v pht trin lun cc r th cp. Nhng th nghim M cho thy Azospirillum c th thay th c 40kg N/ ha/ nm (Smith et al., 1978). Azospirillum cn lm tng sn lng la m Mexico 23-63% (Paredes- Cardona et al., 1988), Argentina 13-30% (Rodriguez-Caceres et al., 1994). Nhng th nghim bc u trn cy bp trng C Lao Dung, Sc Trng, Vit Nam cho thy A. lipoferum HA28 c th gip nng dn tit kim 90N/ha v gip gia tng nng sut bp (Hip et al., 2009).

Mc tiu ca nghin cu ny nhm xc nh kh nng tng hp IAA ca vi khun Azospirillum lipoferum cng nh kho st nh hng ca vi khun ln chiu di r la trong iu kin nh li.

2 PHNG TIN V PHNG PHP

2.1 a im nghin cu

Cc th nghim c tin hnh ti phng th nghim Vi sinh vt, phng Sinh hc Phn t v nh li Vin Nghin cu v Pht trin Cng ngh Sinh hc, Trng i hc Cn Th t thng 01 n thng 04 nm 2009.

2.2 Phng php nghin cu

2.2.1 So snh kh nng tng hp IAA ca mt s dng vi khun Azospirillum lipoferum trong iu kin phng th nghim

Chn ngu nhin 3 dng vi khun Azospirillum lipoferum (R7b1, R8b2 v R29b1) phn lp c t cy la tnh Bc Liu, ng bng Sng Cu Long, Vit Nam. Ba dng vi khun ny c nhn din bng k thut PCR vi cp mi chuyn bit da trn gen nif H v c s dng kho st kh nng tng hp IAA trong iu kin phng th nghim. Th nghim c b tr theo th thc hon ton ngu nhin, 3 ln lp li. Cc dng vi khun ny cng vi cc dng vi khun i chng (Ab 28, Ab10 v Ab39) c nhn mt s trong mi trng NFb lng, khng N, lc nhit phng (28-300C), 200 vng/pht, che kn ng nghim bng bao nylon en trong khi trnh IAA b nh sng phn hu. Xc nh nng IAA do vi khun to ra trong 1, 2, 4, 6 v 8 ngy sau khi chng bng cch ht


134

1,5ml sinh khi vi khun ly tm 5.500 vng/pht trong 10 pht, nh nhng ht 1ml dch trong ca mu sau khi ly tm trn vi 2ml thuc th Salkowski R2 (4,5g Fe2Cl trong 1lt H2SO4 10,8M), mu trong ti 15 pht trc khi o OD bc sng 530nm. Da vo ng chun c nng IAA tng dn: 0, 5, 10, 20, 30, 40 g/ml xc nh lng IAA do vi khun to ra (Eric Glickmann et al., 1995).

2.2.2 Kho st nh hng ca vi khun Azospirillum lipoferum ln chiu di r la

Ht la Mt Bi c kh trng bng cn 5 pht, oxy gi 10% trong 3 pht, ri ngm ht trong nc m 60oC n 70oC trong 30 pht ht trong ti cho ti khi ht nt nanh chun b ny mm. Gieo ht ny mm v thu m sau 2-3 ngy gieo. Chn nhng cy c chiu cao tng i ng u b tr th nghim (Hnh 1).

Hnh 1: Ngm m trong dch vi khun

Dng mi trng NFb lng, khng N nhn mt s 3 dng vi khun Azospirillum lipoferum ( R7b1, R8b2 v R29b1) t mt s 109 CFU/ml.

Th nghim c 4 nghim thc c b tr hon ton ngu nhin vi 4 ln lp li. Nghim thc 1 (nghim thc i chng): m ngm trong nc ct 30 pht. Nghim thc 2, 3, 4 ln lt ngm m ring bit 30 pht trong dch vi khun R7b1, R8b2 hay R29b1. Ct c kh trng nhit t 2 ln, cch ngy 121oC trong 20 pht. Ly c kh trng bng cn 70o. Cn chnh xc 300g ct kh trng cho vo ly nha. Sau , m c trng trong ly vi ct kh trng (Hnh 2).

R29b1

R29b1

R8b2

R8b2

R7b1

R7b1


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Hnh 2: Trng la trong ly vi ct kh trng

Ti la hng ngy bng mi trng khong khng N (Kronzucker et al., 1999) gm (g/l): 3g NaHCO3, 1g NaCl, 1g KH2PO4, 0,5g MgCl2, 1g NH4Cl, 0,1g CaCl2, 0,7g Na2SO4, 1g Na-acetate, 1ml dung dch khong vi lng, 1ml dung dch vitamin v 10ml Sodium ascorbate. o chiu di r cc ngy 7, 14, 21 v 28 sau khi cy.

3 KT QU V THO LUN

3.1 Lng IAA do vi khun tng hp trong mi trng khng c tryptophan

C ba dng vi khun c kho st (R7b1, R8b2 v R29b1) u c kh nng tng hp acid indole-3-acetic (IAA) m khng cn b sung Tryptophan vo mi trng nui vi khun. Lng IAA do cc dng ny to ra cao hn cc dng vi khun i chng. Tuy nhin hm lng IAA tng hp c c s khc nhau gia ba dng, hm lng IAA ny khong 16,656 gIAA/ml n 19,9gIAA/ml (Hnh 3)

0

5

10

15

20

25

Ab28 ab10 ab39 r7b1 r8b2 r29b1

Cc dng vi khun

Nn

g

IA

A Ngy 1

Ngy 2

Ngy 4

Ngy 6

Ngy 8

Hnh 3: Nng IAA do vi khun sn sinh ra trong 8 ngy

Ab28, Ab10 v Ab39: Vi khun Azospirillum lipoferum i chng dng.

R7b1, r8b2 v r29b1: cc vi khun cn xc nh kh nng tng hp IAA.


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Sau mt ngy chng, cc dng vi khun Azospirillum lipoferum sn sinh ra IAA nhng nng cn thp. Sau , nng IAA tip tc tng v tng cao nht ngy th 4 (i vi cc dng Ab39, R8b2 v R29b1) cn cc dng Ab28, Ab10 v R7b1 t nng IAA ti a vo ngy th 6 sau khi chng (Hnh 3).

Hnh 4: IAA do vi khun R29b1 to ra so vi ng chun t 0-30g/ml

Dng R29b1 sinh ra lng IAA cao nht l 19,9 (g/ml) vo ngy th 4 (Hnh 4) v khc bit c ngha so vi cc dng cn li (bng 1). Trong khi , kt qu ca Andriollo et al. (1990) cho thy IAA khong 0,5-18 (g/ml) khi b sung 50 (g/ml) Tryptophan vo mi trng nui vi khun. Cn kt qu ca Patten et al. (2002) khi c b sung Tryptophan vi nng 500 (g/ml) vo mi trng nui vi khun th vi khun sn sinh c lng IAA l 32,72,9 (g/ml/OD600 unit). Khalid et al. (2001) khi phn lp mt s chng vi sinh vt t vng r ca cy la go v la m v th kh nng tng hp IAA ca cc dng vi khun , cho thy lng IAA tng hp c ca cc dng cao nht l 12,1 gIAA/ml. Tuy nhin, cc dng vi khun R7b1, R8b2 v R29b1 s dng trong nghin cu ny sn sinh lng IAA ph hp vi kt qu ca Ral Osvaldo Pedraza et al. (2004), nghin cu cho thy cc dng Azospirillum sp. tng hp c IAA khong 16,538 g IAA/ml.

Bng 1: Lng IAA do cc dng vi khun to ra

Dng vi khun Lng IAA c sinh ra (g/ml)

Ab39

Ab10

Ab28

R7b1

R8b2

R29b1

14,06 f

14,875 e

16,05 d

16,675 c

18,2 b

19,9 a

CV (%) 2,24

Trong cng mt ct nhng s c ch theo sau ging nhau th khc bit khng ngha mc 5% qua php th Duncan.

3.2 nh hng ca vi khun ln chiu di r la trng trong nh li

Sau khi chng vi khun 7 ngy, chiu di r la cc nghim thc khng khc bit vi nhau v khng khc bit so vi i chng v mt thng k (Hnh 5) (Bng 2), c th do vi khun cha thi gian tc ng ln chiu di r la.

30 20 R29b1 10 5 0


137

Hnh 5: nh hng ca vi khun ln r la sau 7 ngy chng

Bng 2: Chiu di r la sau 7 ngy chng vi khun

Nghim thc Chiu di r la (cm)

i chng 4,875 a R7b1 7,375 a

R8b2 7,125 a

R29b1 7, 625 a

CV (%) 29,55


Tuy nhin, chiu di r la c ba nghim thc c chng vi khun khc bit c ngha so vi nghim thc i chng mc ngha 5% vo ngy 14 sau khi chng (Hnh 6), do vi khun c thi gian gip r la pht trin di hn i chng. Kt qu tng t cng c Abbas (2007) tm thy khi r la m trong dch vi khun Azospirillum sp. trong 14 ngy th r ca cc cy ny di hn, c nhiu r ph hn so vi ch trong nc ct.

0

5

10

15

i chng R29b1 R7b1 R8b2

Ch

iu

d

i R

L

a

(cm

)

Hnh 6: nh hng ca vi khun ln chiu di r la 14 ngy sau khi chng

n ngy th 21, nghim thc chng vi khun R29b1 cho chiu di r la cao nht, khc bit c ngha so vi tt c cc nghim thc cn li v gp 2,03 ln so vi i chng. Dng R7b1 v R8b2 cng gip tng chiu di r la, khc bit mc ngha 5% so vi i chng (Hnh 7); do c ba dng vi khun ny u c kh nng tng hp IAA v dng R29b1 to ra lng IAA nhanh hn hai dng cn li nn gip r la di hn. Kt qu ny ph hp vi kt qu ca th nghim kho st

a

a a a

b


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lng IAA sinh ra v c Kolb v Martin (1985) chng minh bng th nghim phun IAA c nng l 10-9g/l vo r ca cy la m kt qu l r cy di hn.

0

5

10

15

20


Ch

iu

d

i R

L

a


n ngy th 28, r la cc nghim thc bn vi khun u c chiu di khc bit so vi i chng (Hnh 8).

0

5

10

15

20

25


Ch

iu

d

i r l

a

(cm

)


Dng R29b1 c chiu di r la gp 2,03 ln so vi i chng. Hai dng R7b1 v R8b2 ln lt gp 1,83 v 1,93 ln so vi i chng. Ngoi ra, s lng r ph cc nghim thc c chng vi khun nhiu hn so vi nghim thc i chng (Hnh 9, Bng 3).

Hnh 9: nh hng ca vi khun ln r la sau 28 ngy chng

b b a

c

b

a a a


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Bng 3: Chiu di r la 28 ngy sau khi chng vi khun

Nghim thc Chiu di r la

i chng 10,25 b R7b1 18,75 a

R8b2 19,75 a

R29b1 20,25 a

CV (%) 13,39


Nh vy, sau 28 ngy chng, cc dng vi khun gip tng chiu di r cy la. c bit, dng R29b1 gip tng chiu di r la ln 2,03 ln so vi i chng. Kt qu ny tng t nh kt qu ca Kolb v Martin (1985) khi phun dch vi khun Azospirillum brasilense FT-326 trn r ca Beta vulgaris lm cho r mc di hn v pht trin c cc r th cp v Amalia et al. (1988) cng chng minh Azospirillum brasilense lm tng chiu di r Panicum miliaceum.

4 KT LUN V NGH

4.1 Kt lun

C ba dng R7b1, R8b2 v R29b1 u c kh nng tng hp IAA m khng cn thm tryptophan vo mi trng nui. Lng IAA nhiu nht c dng R29b1 tng hp l 19,9g/ml.

Chiu di r la cc nghim thc c chng vi khun khc bit so vi i chng mt cch c ngha. Dng R29b1 gip r la di hn 2,03 ln so vi i chng sau 28 ngy chng.

4.2 ngh

Th nghim ngoi ng cn c tin hnh nhiu vng sinh thi khc nhau hng n vic sn xut phn sinh hc cho cy la, xy dng nn nng nghip bn vng, thn thin vi mi trng.

TI LIU THAM KHO

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the isolation of N2-fixing bacteria of the genus Azospirillum. Nitrogen fixation. 48: 347-

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for auxin biosynthesis. Journal of Biological Sciences.18: 750-754.

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Salkowski Reagent for Indolic Compounds produced by Phytopathogenic Bacteria. Appl.

Environ. Microbiol., 61: 793-796.

Glickmann E. and Y. Dessaux. 1995. A critical examination of the Salkowski reagent for

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017.Tran Van Chieu_htt (132-140)

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